Study On The Regulation Mechanism Of Non-coding RNA In The Photosynthetic Hydrogen Production Process Of Chlamydomonas Reinhardtii

To further understand how non-coding RNAs regulate the bio-hydrogen production in the green alga Chlamydomonas reinhardtii, in this study the physiological and biochemical characteristics of two strain transgenic algae were investigated, which have been previously engineered under the control of the heat-inducible promoter in our lab. One referred as “Transformant-miR1166.1” was characterized with the over-expression of C. reinhardtii endogenous microRNA?miR1166.1?, while another referred as “Transformant-amiRNA-D1” was constructed to over produce an artificial microRNA targeting D1 gene?psbA, a key component of photosystem II?. Moreover, for the first time the long non-coding RNA?lncRNA? in C. reinhardti were also screened and identified. More and more attentions have been paid on lncRNAs which may improve C. reinhardtii hydrogen production. The results were as following:1. Regulation mechanism of hydrogen production in Transformant-miR1166.1: after heat shock treatment, the Fv/Fm, Yield, q P and ETR reflecting photosystem activities of transgenic algae decreased by 19.55%, 26.37%, 26.37% and 12.21%, respectively. Interestingly, in vitro hydrogenase activity of miRNA-1166.1 increased by 3.708?mol/mg·Chl·h. Moreover, after heat shock treatment, the content of starch in transgenic algae reduced by 66.71%, while the content of total protein and total fatty acids was found no obviously changed. Finally, the results of qRT-PCR showed that the gene expression of HYDA1, HYDA2 and LHCBM1 was down-regulated by 47.7%, 13.4%, 11.4% respectively, while the gene expression of LHCBM9 increased 101 folds. PSB1 gene expression did not show significant difference between the wild-type CC849 and transgenic algae after heat shock treatment;2. Regulation mechanism of hydrogen production in Transformant-amiRNA-D1: the Fv/Fm, Yield, qP and ETR of transgenic algae decreased by 46.05%, 58.16%, 58.16% and 30.52% respectively after heat shock treatment; Interestingly, in vitro hydrogenase activity of amiRNA-D1 increased by 4.631?mol/mg·Chl·h; Then the content of starch in transformant-amiRNA-D1 reduced by 61.72%, while the content of total protein and total fatty acids was found statistically unchanged. Finally, the genes expression of HYDA1, HYDA2, LHCBM1, PSB1 reduced by 58.6%, 88.6%, 48.9%, 73.4% respectively, while the gene expression of LHCBM9 raised for 90 folds. Compared with the wild-type CC849, these m RNA levels of these genes showed significant difference after heat shock treatment;3. lncRNA screening and identification in C. reinhardti: whole transcriptome strand specific RNA-seq was performed in C. reinhardti, which obtained 1,440 high-confidence lncRNAs, including 936 long intergenic non-coding RNAs?lincRNA?, 310 intronic lncRNA, and 194 antisense lncRNA; C. reinhardtii lncRNA average length was 509 nt, most exons lengths were less than 300 bp, and the numbers of exons were mostly one or two; Interestingly, the differentially expressed and photosynthesis related lncRNAs XLOC₀37917, XLOC₀37244 and XLOC₀37246 had multiple target genes; Further analysis of these 1,440 lnc RNAs with annotated genes in C. reinhardtii showed that the average transcript length, the ORF length and the number of exons are much less than the later respectively; Finally, 21 lncRNAs were randomly selected to confirm their expressions in C. reinhardtii and they responses to sulfur-deprived stress. The result showed expression levels of 18 lncRNA are consistent with the results of RNA-seq sequencing;In conclusion, this study not only analyzed the regulation mechanisms of bio-hydrogen production in transgenic algae, but also identified the long-chain non-coding RNA of C.reinhardtii. miRNA affected its target genes and photosynthesis related genes, while lncRNAs had various relationships with its target genes. To sum up, we should consider using multiple non-coding RNAs to improve hydrogen production in C. reinhardtii.